Nucleic acid mediated electron transfer

ABSTRACT

The present invention provides for the selective covalent modification of nucleic acids with redox active moieties such as transition metal complexes. Electron donor and electron acceptor moieties are covalently bound to the ribose-phosphate backbone of a nucleic acid at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very large distances at extremely fast rates. These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

This is a continuation of application Ser. No. 08/709,263 filed Sep. 6,1996, now U.S. Pat. No. 5,780,234, which is a continuation ofapplication Ser. No. 08/166,036, filed Dec. 10, 1993, now U.S. Pat. No.5,591,578.

FIELD OF THE INVENTION

The present invention is directed to electron transfer via nucleicacids. More particularly, the invention is directed to thesite-selective modification of nucleic acids with electron transfermoieties such as redox active transition metal complexes to produce anew series of biomaterials and to methods of making and using them. Thenovel biomaterials of the present invention may be used as bioconductorsand diagnostic probes.

BACKGROUND OF THE INVENTION

The present invention, in part, relates to methods for thesite-selective modification of nucleic acids with redox active moietiessuch as transition metal complexes, the modified nucleic acidsthemselves, and their uses. Such modified nucleic acids are particularlyuseful as bioconductors and photoactive nucleic acid probes.

The detection of specific nucleic acid sequences is an important toolfor diagnostic medicine and molecular biology research. Gene probeassays currently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

Specificity, in contrast, remains a problem in many currently availablegene probe assays. The extent of molecular complementarity between probeand target defines the specificity of the interaction. Variations in theconcentrations of probes, of targets and of salts in the hybridizationmedium, in the reaction temperature, and in the length of the probe mayalter or influence the specificity of the probe/target interaction.

It may be possible under some limited circumstances to distinguishtargets with perfect complementarity from targets with mismatches,although this is generally very difficult using traditional technology,since small variations in the reaction conditions will alter thehybridization. New experimental techniques for mismatch detection withstandard probes include DNA ligation assays where single pointmismatches prevent ligation and probe digestion assays in whichmismatches create sites for probe cleavage.

Finally, the automation of gene probe assays remains an area in whichcurrent technologies are lacking. Such assays generally rely on thehybridization of a labelled probe to a target sequence followed by theseparation of the unhybridized free probe. This separation is generallyachieved by gel electrophoresis or solid phase capture and washing ofthe target DNA, and is generally quite difficult to automate easily.

The time consuming nature of these separation steps has led to twodistinct avenues of development. One involves the development ofhigh-speed, high-throughput automatable electrophoretic and otherseparation techniques. The other involves the development ofnon-separation homogeneous gene probe assays.

For example, Gen-Probe Inc., (San Diego, Calif.) has developed ahomogeneous protection assay in which hybridized probes are protectedfrom base hydrolysis, and thus are capable of subsequentchemiluminescence. (Okwumabua et al. Res. Microbiol. 143:183 (1992)).Unfortunately, the reliance of this approach on a chemiluminescentsubstrate known for high background photon emission suggests this assaywill not have high specificity. EPO application number 86116652.8describes an attempt to use non-radiative energy transfer from a donorprobe to an acceptor probe as a homogeneous detection scheme. However,the fluorescence energy transfer is greatly influenced by both probetopology and topography, and the DNA target itself is capable ofsignificant energy quenching, resulting in considerable variability.Therefore there is a need for DNA probes which are specific, capable ofdetecting target mismatches, and capable of being incorporated into anautomated system for sequence identification.

As outlined above, molecular biology relies quite heavily on modified orlabelled oligonucleotides for traditional gene probe assays(Oligonucleotide Synthesis: A Practical Approach. Gait et al., Ed., IRLPress: Oxford, UK, 1984; Oligonucleotides and Analogues: A PracticalApproach. Ed. F. Eckstein, Oxford University Press, 1991). As a result,several techniques currently exist for the synthesis of tailored nucleicacid molecules. Since nucleic acids do not naturally contain functionalgroups to which molecules of interest may easily be attached covalently,methods have been developed which allow chemical modification at eitherof the terminal phosphates or at the heterocyclic bases (Dreyer et al.Proc. Natl. Acad. Sci. USA, 1985, 82:968).

For example, analogues of the common deoxyribo- and ribonucleosideswhich contain amino groups at the 2' or 3' position of the sugar can bemade using established chemical techniques. (See Imazawa et al., J. Org.Chem., 1979, 44:2039; Imazawa et al., J. Org. Chem. 43(15):3044 (1978);Verheyden et al., J. Org. Chem. 36(2):250 (1971); Hobbs et al., J. Org.Chem. 42(4):714 (1977)). In addition, oligonucleotides may besynthesized with 2'-5' or 3'-5' phosphoamide linkages (Beaucage et al.,Tetrahedron 49(10):1925 (1992); Letsinger, J. Org. Chem., 35:3800(1970); Sawai, Chem. Lett. 805 (1984); Oligonucleotides and Analogues: APractical Approach, F. Eckstein, Ed. Oxford University Press (1991)).

The modification of nucleic acids has been done for two general reasons:to create nonradioactive DNA markers to serve as probes, and to usechemically modified DNA to obtain site-specific cleavage.

To this end, DNA may be labelled to serve as a probe by altering anucleotide which then serves as a replacement analogue in the nicktranslational resynthesis of double stranded DNA. The chemically alterednucleotides may then provide reactive sites for the attachment ofimmunological or other labels such as biotin. (Gilliam et al., Anal.Biochem. 157:199 (1986)). Another example uses ruthenium derivativeswhich intercalate into DNA to produce photoluminescence under definedconditions. (Friedman et al., J. Am. Chem. Soc. 112:4960 (1990)).

In the second category, there are a number of examples of compoundscovalently linked to DNA which subsequently cause DNA chain cleavage.For example 1,10-phenanthroline has been coupled to single-strandedoligothymidylate via a linker which results in the cleavage of poly-dAoligonucleotides in the presence of Cu²⁺ and 3-mercaptopropionic acid(Francois et al., Biochemistry 27:2272 (1988)). Similar experiments havebeen done for EDTA¹ -Fe(II) (both for double stranded DNA (Boutorin etal., FEBS Lett. 172:43-46 (1986)) and triplex DNA (Strobel et al.,Science 249:73 (1990)), porphyrin-Fe(III) (Le Doan et al.,Biochemistry25:6736-6739 (1986)), and 1,10-phenanthronine-Cu(I) (Chen etal., Proc. Natl. Acad. Sci USA, 83:7147 (1985)), which all result in DNAchain cleavage in the presence of a reducing agent in aerated solutions.A similar example using porphyrins resulted in DNA strand cleavage, andbase oxidation or cross-linking of the DNA under very specificconditions (Le Doan et al., Nucleic Acids Res. 15:8643 (1987)).

Other work has focused on chemical modification of heterocyclic bases.For example, the attachment of an inorganic coordination complex,Fe-EDTA, to a modified internal base resulted in cleavage of the DNAafter hybridization in the presence of dioxygen (Dreyer et al., Proc.Natl. Acad. Sci. USA 82:968 (1985)). A ruthenium compound has beencoupled successfully to an internal base in a DNA octomer, withretention of both the DNA hybridization capabilities as well as thespectroscopic properties of the ruthenium label (Telser et al., J. Am.Chem. Soc. 111:7221 (1989)). Other experiments have successfully addedtwo separate spectroscopic labels to a single double-stranded DNAmolecule (Telser et al., J. Am. Chem. Soc. 111:7226 (1989)).

The study of electron transfer reactions in proteins and DNA has alsobeen explored in pursuit of systems which are capable of long distanceelectron transfer.

To this end, intramolecular electron transfer in protein-proteincomplexes, such as those found in photosynthetic proteins and proteinsin the respiration pathway, has been shown to take place overappreciable distances in protein interiors at biologically significantrates (see Bowler et al., Progress in Inorganic Chemistry: BioinorganicChemistry, Vol. 38, Ed. Stephen J. Lippard (1990). In addition, theselective modification of metalloenzymes with transition metals has beenaccomplished and techniques to monitor electron transfer in thesesystems developed. For example, electron transfer proteins such ascytochrome c have been modified with ruthenium through attachment atseveral histidines and the rate of electron transfer from the heme Fe²⁺to the bound Ru³⁺ measured. The results suggest that electron transfer"tunnel" pathways may exist. (Baum, Chemical & Engineering News, Feb.22, 1993, pages 2023; see also Chang et al., J. Am. Chem. Soc. 113:7056(1991)). In related work, the normal protein insulation, which protectsthe redox centers of an enzyme or protein from nondiscriminatoryreactions with the exterior solvent, was "wired" to transform thesesystems from electrical insulators into electrical conductors (Heller,Acc. Chem. Res. 23:128 (1990)).

There are a few reports of photoinduced electron transfer in a DNAmatrix. In these systems, the electron donors and acceptors are notcovalently attached to the DNA, but randomly associated with the DNA,thus rendering the explicit elucidation and control of thedonor-acceptor system difficult. For example, the intense fluorescenceof certain quaternary diazoaromatic salts is quenched upon intercalationinto DNA or upon exposure to individual mononucleotides, thus exhibitingelectron donor processes within the DNA itself. (Brun et al., J. Am.Chem. Soc. 113:8153 (1991)).

Another example of the difficulty of determining the electron transfermechanism is found in work done with some photoexcitable rutheniumcompounds. Early work suggested that certain ruthenium compounds eitherrandomly intercalate into the nucleotide bases, or bind to the helixsurface. (Purugganan et al., Science 241:1645 (1988)). A recentreference indicates that certain ruthenium compounds do not intercalateinto the DNA (Satyanarayana et al., Biochemistry 31(39):9319 (1992));rather, they bind non-covalently to the surface of the DNA helix.

In these early experiments, various electron acceptor compounds, such ascobalt, chromium or rhodium compounds were added to certainDNA-associated ruthenium electron donor compounds. (Puragganan et al.,Science 241:1645 (1988); Orellana et al., Photochem. Photobiol. 499:54(1991); Brun et al., J. Am. Chem. Soc. 113:8153 (1991);

Davis, Chem.-Biol. Interactions 62:45 (1987); Tomalia et al., Acc. Chem.Res., 24:332 (1991)). Upon addition of these various electron acceptorcompounds, which randomly bind non-covalently to the helix, quenching ofthe photoexcited state through electron transfer was detected. The rateof quenching was dependent on both the individual electron donor andacceptor as well as their concentrations, thus revealing the process asbimolecular.

In one set of experiments, the authors postulate that the more mobilesurface bound donor promotes electron transfer with greater efficiencythan the intercalated species, and suggest that the sugar-phosphatebackbone of DNA, and possibly the solvent medium surrounding the DNA,play a significant role in the electron transport. (Purugganan et al.,Science 241:1645 (1988)). In other work, the authors stress thedependence of the rate on the mobility of the donor and acceptor andtheir local concentrations, and assign the role of the DNA to beprimarily to facilitate an increase in local concentration of the donorand acceptor species on the helix. (Orellana et al., supra).

In another experiment, an electron donor was reportedly randomlyintercalated into the stack of bases of DNA, while the acceptor wasrandomly associated with the surface of the DNA. The rate of electrontransfer quenching indicated a close contact of the donor and theacceptor, and the system also exhibits enhancement of the rate ofelectron transfer with the addition of salt to the medium. (Fromherz etal., J. Am. Chem. Soc. 108:5361 (1986)).

In all of these experiments, the rate of electron transfer fornon-covalently bound donors and acceptors is several orders of magnitudeless than is seen in free solution.

An important stimulus for the development of long distance electrontransfer systems is the creation of synthetic light harvesting systems.Work to date suggests that an artificial light harvesting systemcontains an energy transfer complex, an energy migration complex, anelectron transfer complex and an electron migration complex (for atopical review of this area, see Chemical & Engineering News, Mar. 15,1993, pages 38-48). Two types of molecules have been tried: a) longorganic molecules, such as hydrocarbons with covalently attachedelectron transfer species, or DNA, with intercalated, partiallyintercalated or helix associated electron transfer species, and b)synthetic polymers.

The long organic molecules, while quite rigid, are influenced by anumber of factors, which makes development difficult. These factorsinclude the polarity and composition of the solvent, the orientation ofthe donor and acceptor groups, and the chemical character of either thecovalent linkage or the association of the electron transfer species tothe molecule.

The creation of acceptable polymer electron transfer systems has beendifficult because the available polymers are too flexible, such thatseveral modes of transfer occur. Polymers that are sufficiently rigidoften significantly interfere with the electrontransfer mechanism or arequite difficult to synthesize.

Thus the development of an electron transfer system which issufficiently rigid, has covalently attached electron transfer species atdefined intervals, is easy to synthesize and does not appreciablyinterfere with the electron transfer mechanism would be useful in thedevelopment of artificial light harvesting systems.

In conclusion, the random distribution and mobility of the electrondonor and acceptor pairs, coupled with potential short distances betweenthe donor and acceptor, the loose and presumably reversible associationof the donors and acceptors, the reported dependence on solvent andbroad putative electron pathways, and the disruption of the DNAstructure of intercalated compounds rendering normal base pairingimpossible all serve as pronounced limitations of long range electrontransfer in a DNA matrix. Therefore, a method for the production ofrigid, covalent attachment of electron donors and acceptors to provideminimal perturbations of the nucleic acid structure and retention of itsability to base pair normally, is desirable. The present inventionserves to provide such a system, which allows the development of novelbioconductors and diagnostic probes.

SUMMARY OF THE INVENTION

The present invention provides for the selective modification of nucleicacids at specific sites with redox active moieties such as transitionmetal complexes. An electron donor and/or electron acceptor moiety arecovalently bound preferably along the ribose-phosphate backbone of thenucleic acid at predetermined positions. The resulting complexesrepresent a series of new derivatives that are biomolecular templatescapable of transferring electrons over very large distances at extremelyfast rates. These complexes possess unique structural features whichenable the use of an entirely new class of bioconductors and diagnosticprobes.

Accordingly, it is an object of the invention to provide a singlestranded nucleic acid which has both an electron donor moiety and anelectron acceptor moiety covalently attached thereto. These moieties areattached through the ribose phosphate or analogous backbone of thenucleic acid. The single stranded nucleic acid is capable of hybridizingto a complementary target sequence in a single stranded nucleic acid,and transferring electrons between the donor and acceptor.

It is a further object of the present invention to provide for a nucleicacid probe which can detect base-pair mismatches. In this embodiment,the single stranded nucleic acid with a covalently attached electrondonor and electron acceptor moiety is hybridized to a complementarytarget sequence in a single stranded nucleic acid. When the region ofhybridization contains at least one base pair mismatch, the rate ofelectron transfer between the donor moiety and the acceptor moiety isdecreased or eliminated, as compared to when there is perfectcomplementarity between the probe and target sequence.

It is an additional object of the present invention to provide a complexwhich contains a first single stranded nucleic acid with at least oneelectron donor moiety and a second single stranded nucleic acid with atleast one electron acceptor moiety. As with the other embodiments of thepresent invention, the moieties are covalently linked to theribose-phosphate backbone of the nucleic acids.

In one aspect of the present invention, the first and second singlestranded nucleic acids are capable of hybridizing to each other to forma double stranded nucleic acid, and of transferring electrons betweenthe electron donor moiety and the electron acceptor moiety.

In another aspect of the present invention, a target sequence in asingle stranded nucleic acid comprises at least first and second targetdomains, which are directly adjacent to one another. The first singlestranded nucleic acid hybridizes to the first target domain and thesecond single stranded nucleic acid hybridizes to the second targetdomain, such that the first and second single stranded nucleic acids areadjacent to each other. This resulting hybridization complex is capableof transferring electrons between the electron donor moiety and theelectron acceptor moiety on the first and second nucleic acids.

In another aspect of the present invention, a target sequence in asingle stranded nucleic acid comprises a first target domain, anintervening target domain, and a second target domain. The interveningtarget domain comprises one or more nucleotides. The first and secondsingle stranded nucleic acids hybridize to the first and second targetdomains. An intervening nucleic acid comprising one or more nucleotideshybridizes to the target intervening domain such that electrons arecapable of being transferred between the electron donor moiety and theelectron acceptor moiety on the first and second nucleic acids.

The invention also provides for a method of making a single strandednucleic acid containing an electron transfer moiety covalently attachedto the 5' terminus of the nucleic acid. The method comprisesincorporating a modified nucleotide into a growing nucleic acid at the5' position to form a modified single stranded nucleic acid. Themodified single stranded nucleic acid is then hybridized with acomplementary single stranded nucleic acid to form a double strandednucleic acid. The double stranded nucleic acid is reacted with anelectron transfer moiety such that the moiety is covalently attached tothe modified single stranded nucleic acid. The modified single strandednucleic acid containing the electron transfer moiety is separated fromthe complementary unmodified single stranded nucleic acid.

The present invention also provides a method for making a singlestranded nucleic acid containing an electron transfer moiety covalentlyattached to an internal nucleotide. The method comprises creating anucleotide dimer joined by a phosphoramide bond and incorporating saidnucleotide dimer into a growing nucleic acid to form a modified singlestranded nucleic acid. The modified single stranded nucleic acid is thenhybridized with a complementary single stranded nucleic acid to form adouble stranded nucleic acid. The double stranded nucleic acid isreacted with an electron transfer moiety such that the moiety iscovalently attached to the modified single stranded nucleic acid. Themodified single stranded nucleic acid containing the electron transfermoiety is separated from the complementary unmodified single strandednucleic acid.

Another aspect of the present invention provides a method of detecting atarget sequence. The method comprises creating a single stranded nucleicacid with an electron donor moiety and an electron acceptor moietycovalently attached. The single stranded nucleic acid containing theelectron transfer moieties is then hybridized to the target sequence,and an electron transfer rate determined between the electron donor andthe electron acceptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H illustrate possible orientations of electron donor (EDM) andelectron acceptor (EAM) moieties on a single stranded nucleic acid.

FIGS. 2A1-2A9 and 2B1-2B9 illustrate orientations of electron transfermoieties EDM and EAM on two adjacent single stranded nucleic acids.These orientations also apply when the two probes are separated by anintervening sequence.

FIG. 3 illustrates a series of amino-modified nucleoside precursorsprior to incorporation into an oligonucleotide.

FIGS. 4A and 4B depict the structure of electron transfer moieties. FIG.4A depicts the general formula of a representative class of electrondonors and acceptors. FIG. 4B depicts a specific example of a rutheniumelectron transfer moiety using bisbipyridine and imidazole as theligands.

DETAILED DESCRIPTION

Unless otherwise stated, the term "nucleic acid" or "oligonucleotide" orgrammatical equivalents herein means at least two nucleotides covalentlylinked together. A nucleic acid of the present invention will generallycontain phosphodiester bonds, although in some cases, as outlined below,a nucleic acid may have an analogous backbone, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970)),phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), or peptide nucleic acid linkages (see Egholm,J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993)). The nucleic acids maybe single stranded or double stranded, as specified. The nucleic acidmay be DNA, RNA or a hybrid, where the nucleic acid contains anycombination of deoxyribo- and ribo-nucleotides, and any combination ofuracil, adenine, thymine, cytosine and guanine. In some instances, e.g.in the case of an "intervening nucleic acid", the term nucleic acidrefers to one or more nucleotides.

The terms "electron donor moiety", "electron acceptor moiety", and"electron transfer moieties" or grammatical equivalents herein refers tomolecules capable of electron transfer under certain conditions. It isto be understood that electron donor and acceptor capabilities arerelative; that is, a molecule which can lose an electron under certainexperimental conditions will be able to accept an electron underdifferent experimental conditions. Generally, electron transfer moietiescontain transition metals as components, but not always.

The term "target sequence" or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,mRNA, or others. It may be any length, with the understanding thatlonger sequences are more specific. Generally speaking, this term willbe understood by those skilled in the art.

The probes of the present invention are designed to be complementary tothe target sequence, such that hybridization of the target sequence andthe probes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence.

The terms "first target domain" and "second target domain" orgrammatical equivalents herein means two portions of a target sequencewithin a nucleic acid which is under examination. The first targetdomain may be directly adjacent to the second target domain, or thefirst and second target domains may be separated by an interveningtarget domain. The terms "first" and "second" are not meant to confer anorientation of the sequences with respect to the 5'-3' orientation ofthe target sequence. For example, assuming a 5'-3' orientation of thecomplementary target sequence, the first target domain may be locatedeither 5' to the second domain, or 3' to the second domain.

The present invention is directed, in part, to the site-selectivemodification of nucleic acids with redox active moieties such astransition metal complexes for the preparation of a new series ofbiomaterials capable of long distance electron transfer through anucleic acid matrix. The present invention provides for the preciseplacement of electron transfer donor and acceptor moieties atpredetermined sites on a single stranded or double stranded nucleicacid. In general, electron transfer between electron donor and acceptormoieties in a double helical nucleic acid does not occur at anappreciable rate unless nucleotide base pairing exists in the sequencebetween the electron donor and acceptor in the double helical structure.

This differential in the rate of electron transfer forms the basis of autility of the present invention for use as probes. In the system of thepresent invention, where electron transfer moieties are covalently boundto the backbone of a nucleic acid, the electrons putatively travel viathe π-orbitals of the stacked base pairs of the double stranded nucleicacid. The electron transfer rate is dependent on several factors,including the distance between the electron donor-acceptor pair, thefree energy (ΔG) of the reaction, the reorganization energy (λ), thecontribution of the intervening medium, the orientation and electroniccoupling of the donor and acceptor pair, and the hydrogen bondingbetween the bases. The latter confers a dependence on the actual nucleicacid sequence, since A-T pairs contain one less hydrogen bond than C-Gpairs. However, this sequence dependence is overshadowed by thedetermination that there is a measurable difference between the rate ofelectron transfer within a DNA base-pair matrix, and the rate throughthe ribose-phosphate backbone, the solvent or other electron tunnels.This rate differential is thought to be at least several orders ofmagnitude, and may be as high as four orders of magnitude greaterthrough the stacked nucleotide bases as compared to other electrontransfer pathways. Thus the presence of double stranded nucleic acids,for example in gene probe assays, can be determined by comparing therate of electron transfer for the unhybridized probe with the rate forhybridized probes.

In one embodiment, the present invention provides for novel gene probes,which are useful in molecular biology and diagnostic medicine. In thisembodiment, single stranded nucleic acids having a predeterminedsequence and covalently attached electron donor and electron acceptormoieties are synthesized. The sequence is selected based upon a knowntarget sequence, such that if hybridization to a complementary targetsequence occurs in the region between the electron donor and theelectron acceptor, electron transfer proceeds at an appreciable anddetectable rate. Thus, the present invention has broad general use, as anew form of labelled gene probe. In addition, since detectable electrontransfer in unhybridized probes is not appreciable, the probes of thepresent invention allow detection of target sequences without theremoval of unhybridized probe. Thus, the present invention is uniquelysuited to automated gene probe assays.

The present invention also finds use as a unique methodology for thedetection of mutations in target nucleic acid sequences. As a result, ifa single stranded nucleic acid containing electron transfer moieties ishybridized to a target sequence with a mutation, the resultingperturbation of the base pairing of the nucleotides will measurablyaffect the electron transfer rate. This is the case if the mutation is asubstitution, insertion or deletion. Accordingly, the present inventionprovides for the detection of mutations in target sequences.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

In an alternate embodiment double stranded nucleic acids have covalentlyattached electron donor and electron acceptor moieties on oppositestrands. Such nucleic acids are useful to detect successful geneamplification in polymerase chain reactions (PCR). For example, if oneof the two PCR primers contains a 5' terminally attached electron donor,and the other contains a 5' terminally attached electron acceptor,several rounds of PCR will generate doubly labeled double strandedfragments (occasionally referred to as "amplicons"). After appropriatephotoinduction, the detection of electron transfer provides anindication of the successful amplification of the target sequence ascompared to when no amplification occurs. A particular advantage of thepresent invention is that the separation of the single stranded primersfrom the amplified double stranded DNA is not necessary, as outlinedabove for probe sequences which contain electron transfer moieties.

In another embodiment the present invention provides for double strandednucleic acids with covalently attached electron donor and electronacceptor moieties to serve as bioconductors or "molecular wire". Theelectron transport may occur over distances up to and in excess of 28 Åper electron donor and acceptor pair. In addition, the rate of electrontransfer is very fast, even though dependent on the distance between theelectron donor and acceptor moieties. By modifying the nucleic acid inregular intervals with electron donor and/or electron acceptor moieties,it may be possible to transport electrons over long distances, thuscreating bioconductors. These bioconductors are useful in a large numberof applications, including traditional applications for conductors suchas mediators for electrochemical reactions and processes.

In addition, these bioconductors may be useful as probes forphotosynthesis reactions as well as in the construction of syntheticlight harvesting systems. The current models for the electron transfercomponent of an artificial light harvesting system have severalproblems, as outlined above, including a dependence on solvent polarityand composition, and a lack of sufficient rigidity without arduoussynthesis. Thus the present invention is useful as both a novel form ofbioconductor as well as a novel gene probe.

In addition, the present invention provides a novel method for the sitespecific addition to the ribose-phosphate backbone of a nucleic acid ofelectron donor and electron acceptor moieties to a previously modifiednucleotide.

In one embodiment, the electron donor and acceptor moieties are added tothe 3' and/or 5' termini of the nucleic acid. In alternativeembodiments, the electron donor and acceptor moieties are added to thebackbone of one or more internal nucleotides, that is, any nucleotidewhich is not the 3' or 5' terminal nucleotide. In a further embodiment,the electron donor and acceptor moieties are added to the backbone ofboth internal and terminal nucleotides.

In a preferred embodiment, the transition metal electron transfermoieties are added through a procedure which utilizes modifiednucleotides, preferably amino-modified nucleotides. In this embodiment,the electron transfer moieties are added to the sugar phosphate backbonethrough the nitrogen group in phosphoramide linkages. The modifiednucleotides are then used to site-specifically add a transition metalelectron transfer moiety, either to the 3' or 5' termini of the nucleicacid, or to any internal nucleotide.

Molecular mechanics calculations indicate that perturbations due to themodification of the terminal nucleotides of nucleic acids are minimaland Watson-Crick base pairing is not disrupted (unpublished data usingBiograf from Molecular Simulations Inc., San Diego, Calif.).Accordingly, in one embodiment, modified nucleotides are used to add anelectron transfer moiety to the 5' terminus of a nucleic acid. In thisembodiment, the 2' position of the ribose of the deoxyribo- orribonucleoside is modified prior to the addition of the electrontransfer species, leaving the 3' position of the ribose unmodified forsubsequent chain attachment. In a preferred embodiment, an amino groupis added to the 2' carbon of the sugar using established chemicaltechniques. (Imazawa et al., J. Org. Chem., 44:2039 (1979); Hobbs etal., J. Org. Chem. 42(4):714 (1977); Verheyden et al. J. Org. Chem.36(2):250 (1971)).

Once the modified nucleotides are prepared, protected and activated,they may be incorporated into a growing oligonucleotide by standardsynthetic techniques (Gait, oligonucleotide Synthesis: A PracticalApproach, IRL Press, Oxford, UK 1984; Eckstein) as the 5' terminalnucleotide. This method therefore allows the addition of a transitionmetal electron transfer moiety to the 5' terminus of a nucleic acid.

In an alternative embodiment, the 3' terminal nucleoside is modified inorder to add a transition metal electron transfer moiety. In thisembodiment, the 3' nucleoside is modified at either the 2' or 3' carbonof the ribose sugar. In a preferred embodiment, an amino group is addedto the 2' or 3' carbon of the sugar using established chemicaltechniques (Imazawa et al., J. Org. Chem., 44:2039 (1979); Hobbs et al.,J. Org. Chem. 42(4):714 (1977); Verheyden et al. J. Org. Chem. 36(2):250(1971)).

The above procedures are applicable to both DNA and RNA derivatives asshown in FIG. 3.

The amino-modified nucleotides made as described above are converted tothe 2' or 3' modified nucleotide triphosphate form using standardbiochemical methods (Fraser et al., Proc. Natl. Acad. Sci. USA, 4:2671(1973)). One or more modified nucleosides are then attached at the 3'end using standard molecular biology techniques such as with the use ofthe enzyme DNA polymerase I or terminal deoxynucleotidyltransferase(Ratliff, Terminal deoxynucleotidyltransferase. In The Enzymes, Vol 14A.P. D. Boyer ed. pp 105-118. Academic Press, San Diego, Calif. 1981).

In other embodiments, the transition metal electron transfer moiety ormoieties are added to the middle of the nucleic acid, i.e. to aninternal nucleotide. This may be accomplished in three ways.

In a preferred embodiment, an oligonucleotide is amino-modified at the5' terminus as described above. In this embodiment, oligonucleotidesynthesis simply extends the 5' end from the amino-modified nucleotideusing standard techniques. This results in an internally amino modifiedoligonucleotide.

In an alternate embodiment, electron transfer moieties are added to thebackbone at a site other than ribose. For example, phosphoramide ratherthan phosphodiester linkages can be used as the site for transitionmetal modification. These transition metals serve as the donors andacceptors for electron transfer reactions. While structural deviationsfrom native phosphodiester linkages do occur and have been studied usingCD and NMR (Heller, Acc. Chem. Res. 23:128 (1990); Schuhmann et al.

J. Am. Chem. Soc. 113:1394 (1991)), the phosphoramidite internucleotidelink has been reported to bind to complementary polynucleotides and isstable (Beaucage et al., supra, and references therein; Letsinger,supra; Sawai, supra; Jager, Biochemistry 27:7237 (1988)). In thisembodiment, dimers of nucleotides are created with phosphoramidelinkages at either the 2'-5' or 3'-5' positions. A preferred embodimentutilizes the 3'-5' position for the phosphoramide linkage, such thatstructural disruption of the subsequent Watson-Crick basepairing isminimized. These dimer units are incorporated into a growingoligonucleotide chain, as above, at defined intervals, as outlinedbelow.

It should be noted that when using the above techniques for themodification of internal residues it is possible to create a nucleicacid that has an electron transfer species on the next-to-last 3'terminal nucleotide, thus eliminating the need for the extra stepsrequired to produce the 3' terminally labelled nucleotide.

In a further embodiment for the modification of internal residues, 2' or3' modified nucleoside triphosphates are generated using the techniquesdescribed above for the 3' nucleotide modification. The modifiednucleosides are inserted internally into nucleic acid using standardmolecular biological techniques for labelling DNA and RNA. Enzymes usedfor said labelling include DNA polymerases such as polymerase I, T4 DNApolymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptaseand RNA polymerases such as E. coli RNA polymerase or the RNApolymerases from phages SP6, T7 or T3 (Short Protocols in MolecularBiology, 1992. Ausubel et al. Ed. pp 3.11-3.30).

In a preferred embodiment, the electron donor and acceptor moieties areattached to the modified nucleotide by methods which utilize a uniqueprotective hybridization step. In this embodiment, the modified singlestrand nucleic acid is hybridized to an unmodified complementarysequence. This blocks the sites on the heterocyclic bases that aresusceptible to attack by the transition metal electron transfer species.The exposed amine or other ligand at the 2' or 3' position of theribose, the phosphoramide linkages, or the other linkages useful in thepresent invention, are readily modified with a variety of transitionmetal complexes with techniques readily known in the art (see forexample Millet et al, in Metals in Biological Systems, Sigel et al. Ed.Vol. 27, pp 223-264, Marcell Dekker Inc. New York, 1991 and Durham, etal. in ACS Advances in Chemistry Series, Johnson et al. Eds., Vol. 226,pp 180-193, American Chemical Society, Washington D.C.; and Meade etal., J. Am. Chem. Soc. 111:4353 (1989)). After successful addition ofthe desired metal complex, the modified duplex nucleic acid is separatedinto single strands using techniques well known in the art.

In a preferred embodiment, single stranded nucleic acids are made whichcontain one electron donor moiety and one electron acceptor moiety. Theelectron donor and electron acceptor moieties may be attached at eitherthe 5' or 3' end of the single stranded nucleic acid. Alternatively, theelectron transfer moieties may be attached to internal nucleotides, orone to an internal nucleotide and one to a terminal nucleotide. Itshould be understood that the orientation of the electron transferspecies with respect to the 5'-3' orientation of the nucleic acid is notdeterminative. Thus, as outlined in FIG. 1, any combination of internaland terminal nucleotides may be utilized in this embodiment.

In an alternate preferred embodiment, single stranded nucleic acids withat least one electron donor moiety and at least one electron acceptormoiety are used to detect mutations in a complementary target sequence.A mutation, whether it be a substitution, insertion or deletion of anucleotide or nucleotides, results in incorrect base pairing in ahybridized double helix of nucleic acid. Accordingly, if the path of anelectron from an electron donor moiety to an electron acceptor moietyspans the region where the mismatch lies, the electron transfer will beeliminated or reduced such that a change in the relative rate will beseen. Therefore, in this embodiment, the electron donor moiety isattached to the nucleic acid at a 5' position from the mutation, and theelectron acceptor moiety is attached at a 3' position, or vice versa.

In this embodiment it is also possible to use an additional label on themodified single stranded nucleic acid to detect hybridization wherethere is one or more mismatches. If the complementary target nucleicacid contains a mutation, electron transfer is reduced or eliminated. Toact as a control, the modified single stranded nucleic acid may beradio- or fluorescently labeled, such that hybridization to the targetsequence may be detected, according to traditional molecular biologytechniques. This allows for the determination that the target sequenceexists but contains a substitution, insertion or deletion of one or morenucleotides. Alternatively, single stranded nucleic acids with at leastone electron donor moiety and one electron acceptor moiety whichhybridize to regions with exact matches can be used as a controls forthe presence of the target sequence.

It is to be understood that the rate of electron transfer through adouble stranded nucleic acid helix depends on the nucleotide distancebetween the electron donor and acceptor moieties. Longer distances willhave slower rates, and consideration of the rates will be a parameter inthe design of probes and bioconductors. Thus, while it is possible tomeasure rates for distances in excess of 100 nucleotides, a preferredembodiment has the electron donor moiety and the electron acceptormoiety separated by at least 3 and no more than 100 nucleotides. Morepreferably the moieties are separated by 8 to 64 nucleotides, with 15being the most preferred distance.

In addition, it should be noted that certain distances may allow theutilization of different detection systems. For example, the sensitivityof some detection systems may allow the detection of extremely fastrates; i.e. the electron transfer moieties may be very close together.Other detection systems may require slightly slower rates, and thusallow the electron transfer moieties to be farther apart.

In an alternate embodiment, a single stranded nucleic acid is modifiedwith more than one electron donor or acceptor moiety. For example, toincrease the signal obtained from these probes, or decrease the requireddetector sensitivity, multiple sets of electron donor-acceptor pairs maybe used.

As outlined above, in some embodiments different electron transfermoieties are added to a single stranded nucleic acid. For example, whenan electron donor moiety and an electron acceptor moiety are to beadded, or several different electron donors and electron acceptors, thesynthesis of the single stranded nucleic acid proceeds in several steps.First partial nucleic acid sequences are made, each containing a singleelectron transfer species, i.e. either a single transfer moiety orseveral of the same transfer moieties, using the techniques outlinedabove. Then these partial nucleic acid sequences are ligated togetherusing techniques common in the art, such as hybridization of theindividual modified partial nucleic acids to a complementary singlestrand, followed by ligation with a commercially available ligase.

In a preferred embodiment, single stranded nucleic acids are made whichcontain one electron donor moiety or one electron acceptor moiety. Theelectron donor and electron acceptor moieties are attached at either the5' or 3' end of the single stranded nucleic acid. Alternatively, theelectron transfer moiety is attached to an internal nucleotide.

It is to be understood that different species of electron donor andacceptor moieties may be attached to a single stranded nucleic acid.Thus, more than one type of electron donor moiety or electron acceptormoiety may be added to any single stranded nucleic acid.

In a preferred embodiment, a first single stranded nucleic acid is madewith on or more electron donor moieties attached. A second singlestranded nucleic acid has one or more electron acceptor moietiesattached. In this embodiment, the single stranded nucleic acids are madefor use as probes for a complementary target sequence. In oneembodiment, the complementary target sequence is made up of a firsttarget domain and a second target domain, where the first and secondsequences are directly adjacent to one another. In this embodiment, thefirst modified single stranded nucleic acid, which contains onlyelectron donor moieties or electron acceptor moieties but not both,hybridizes to the first target domain, and the second modified singlestranded nucleic acid, which contains only the corresponding electrontransfer species, binds to the second target domain. The relativeorientation of the electron transfer species is not important, asoutlined in FIG. 2, and the present invention is intended to include allpossible orientations.

In the design of probes comprised of two single stranded nucleic acidswhich hybridize to adjacent first and second target sequences, severalfactors should be considered. These factors include the distance betweenthe electron donor moiety and the electron acceptor moiety in thehybridized form, and the length of the individual single strandedprobes. For example, it may be desirable to synthesize only 5'terminally labelled probes. In this case, the single stranded nucleicacid which hybridizes to the first sequence may be relatively short,such that the desirable distance between the probes may be accomplished.For example, if the optimal distance between the electron transfermoieties is 15 nucleotides, then the first probe may be 15 nucleotideslong.

In one aspect of this embodiment, the two single stranded nucleic acidswhich have hybridized to the adjacent first and second target domainsare ligated together prior to the electron transfer reaction. This maybe done using standard molecular biology techniques utilizing a DNAligase, such as T4 DNA ligase.

In an alternative embodiment, the complementary target sequence willhave a first target domain, an intervening target domain, and a secondtarget domain. In this embodiment, the first modified single strandednucleic acid, which contains only electron donor moieties or electronacceptor moieties but not both, hybridizes to the first target domain,and the second modified single stranded nucleic acid, which containsonly the corresponding electron transfer species, binds to the secondtarget domain. When an intervening single stranded nucleic acidhybridizes to the intervening target sequence, electron transfer betweenthe donor and acceptor is possible. The intervening sequence may be anylength, and may comprise a single nucleotide. Its length, however,should take into consideration the desirable distances between theelectron donor and acceptor moieties on the first and second modifiednucleic acids. Intervening sequences of lengths greater than 14 aredesirable, since the intervening sequence is more likely to remainhybridized to form a double stranded nucleic acid if longer interveningsequences are used. The presence or absence of an intervening sequencecan be used to detect insertions and deletions.

In one aspect of this embodiment, the first single stranded nucleic acidhybridized to the first target domain, the intervening nucleic acidhybridized to the intervening domain, and the second single strandednucleic acid hybridized to the second target domain, may be ligatedtogether prior to the electron transfer reaction. This may be done usingstandard molecular biology techniques. For example, when the nucleicacids are DNA, a DNA ligase, such as T4 DNA ligase can be used.

The complementary target single stranded nucleic acid of the presentinvention may take many forms. For example, the complementary targetsingle stranded nucleic acid sequence may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. One skilled in theart of molecular biology would understand how to construct useful probesfor a variety of target sequences using the present invention.

In one embodiment, two single stranded nucleic acids with covalentlyattached electron transfer moieties have complementary sequences, suchthat they can hybridize together to form a bioconductor. In thisembodiment, the hybridized duplex is capable of transferring at leastone electron from the electron donor moiety to the electron acceptormoiety. In a preferred embodiment, the individual single strandednucleic acids are aligned such that they have blunt ends; in alternativeembodiments, the nucleic acids are aligned such that the double helixhas cohesive ends. In either embodiment, it is preferred that there beuninterrupted double helix base-pairing between the electron donormoiety and the electron acceptor moiety, such that electrons may travelthrough the stacked base pairs.

In one bioconductor embodiment, the double stranded nucleic acid has onesingle strand nucleic acid which carries all of the electron transfermoieties. In another embodiment, the electron transfer moieties may becarried on either strand, and in any orientation. For example, onestrand may carry only electron donors, and the other only electronacceptors or both strands may carry both.

In one embodiment, the double stranded nucleic acid may have differentelectron transfer moieties covalently attached in a fixed orientation,to facilitate the long range transfer of electrons. This type of systemtakes advantage of the fact that electron transfer species may act asboth electron donors and acceptors depending on their oxidative state.Thus, an electron donor moiety, after the loss of an electron, may actas an electron acceptor, and vice versa. Thus, electron transfermoieties may be sequentially oriented on either strand of the doublestranded nucleic acid such that directional transfer of an electron oververy long distances may be accomplished. For example, a double strandednucleic acid could contain a single electron donor moiety at one end andelectron acceptor moieties, of the same or different composition,throughout the molecule. A cascade effect of electron transfer could beaccomplished in this manner, which may result in extremely long rangetransfer of electrons.

The choice of the specific electron donor and acceptor pairs will beinfluenced by the type of electron transfer measurement used; for areview, see Winkler et al., Chem. Rev. 92:369-379 (1992). When along-lived excited state can be prepared on one of the redox sites,direct measurement of the electron transfer rate after photoinductioncan be measured, using for example the flash-quench method of Chang etal., J. Amer. Chem. Soc. 113:7057 (1991). In this preferred embodiment,the excited redox site, being both a better acceptor and donor than theground-state species, can transfer electrons to or from the redoxpartner. An advantage of this method is that two electron transfer ratesmay be measured: the photoinduced electron transfer rates and thermalelectron-hole recombination reactions . Thus differential rates may bemeasured for hybridized nucleic acids with perfect complementarity andnucleic acids with mismatches.

In alternative embodiments, neither redox site has a long lived excitedstate, and electron transfer measurements depend upon bimoleculargeneration of a kinetic intermediate. For a review, see Winkler et al.,supra. This intermediate then relaxes to the thermodynamic product viaintramolecular electron transfer using a quencher, as seen below:##EQU1## The upper limit of measurable intramolecular electron transferrates using this method is about 10⁴ per second.

Alternative embodiments use the pulse-radiolytic generation of reducingor oxidizing radicals, which inject electrons into a donor or removeelectrons from a donor, as reviewed in Winkler et al., supra.

Electron transfer will be initiated using electrical, electrochemical,photon (including laser) or chemical activation of the electron transfermoieties. These events are detected by changes in transient absorptionor by fluorescence or phosphorescence or chemiluminescence of theelectron transfer moieties.

In the preferred embodiment, electron transfer occurs afterphotoinduction with a laser. In this embodiment, electron donor moietiesmay, after donating an electron, serve as electron acceptors undercertain circumstances. Similarly, electron acceptor moieties may serveas electron donors under certain circumstances.

In a preferred embodiment, DNA is modified by the addition of electrondonor and electron acceptor moieties. In an alternative embodiment, RNAis modified. In a further embodiment, a double stranded nucleic acid foruse as a bioconductor will contain some deoxyribose nucleotides, someribose nucleotides, and a mixture of adenosine, thymidine, cytosine,guanine and uracil bases.

In accordance with a further aspect of the invention, the preferredformulations for donors and acceptors will possess a transition metalcovalently attached to a series of ligands and further covalentlyattached to an amine group as part of the ribose ring (2' or 3'position) or to a nitrogen or sulfur atom as part of a nucleotide dimerlinked by a peptide bond, phosphoramidate bond, phosphorothioate bond,phosphorodithioate bond or O-methyl phosphoramidate bond.

A general formula is representative of a class of donors and acceptorsthat may be employed is shown in FIG. 4A. In this figure, M may be Cd,Mg, Cu, Co, Pd, Zn, Fe, Ru with the most preferred being ruthenium. Thegroups R¹, R², R³, R⁴, and R⁵ may be any coordinating ligand that iscapable of covalently binding to the chosen metal and may includeligands such as NH₃, pyridine, isonicotinamide, imidazole, bipyridine,and substituted derivative of bipyridine, phenanthrolines andsubstituted derivatives of phenanthrolines, porphyrins and substitutedderivatives of the porphyrin family. The structure of a rutheniumelectron transfer species using bisbipyridine and imidazole as theligands is shown in FIG. 4B. Specific examples of useful electrontransfer complexes include, but are not limited to, those shown in Table1.

                  TABLE 1                                                         ______________________________________                                        Donors             Acceptors                                                  ______________________________________                                        Ru (bpy).sub.2 im-NH.sub.2 -U                                                                    Ru (NH.sub.3).sub.5 -NH.sub.2 -U                             Ru (bpy).sub.2 im-NH.sub.2 -U Ru (NH.sub.3).sub.4 py-NH.sub.2 -U                                Ru (bpy).sub.2 im-NH.sub.2 -U Ru (NH.sub.3).sub.4                            im-NH.sub.2 -U                                             ______________________________________                                         Where:                                                                        Ru = ruthenium                                                                bpy = bisbipyridine                                                           im = imidazole                                                                py = pyridine                                                            

It is to be understood that the number of possible electron donormoieties and electron acceptor moieties is very large, and that oneskilled in the art of electron transfer compounds will be able toutilize a number of compounds in the present invention.

In an alternate embodiment, one of the electron transfer moieties may bein the form of a solid support such as an electrode. When the otherelectron transfer moiety is in solution the system is referred to as aheterogenous system as compared to a homogenous system where bothelectron donor and electron transfer moities are in the same phase.

The techniques used in this embodiment are analogos to the wiring ofproteins to an electrode except that the nucleic acids of the presentinvention are used rather than a redox protein (see for example Gregg etal., J. Phys. Chem. 95:5970 (1991); Heller et al., Sensors and ActuatorsR., 13-14:180 (1993); and Pishko et al., Anal. Chem., 63:2268 (1991)).In this embodiment, it is preferred that a redox polymer such as apoly-(vinylpyridine) complex of Os(bpy)₂ Cl be cross-linked with anepoxide such as diepoxide to form a redox-conducting epoxide cementwhich is capable of strongly binding to electrodes made of conductivematerial such as gold, vitreous carbon, graphite, and other conductivematerials. This strong attachment is included in the definition of"covalently attached" for the purposes of this embodiment. The epoxidecross-linking polymer is then reacted with, for example, an exposedamine, such as the amine of an amino-modified nucleic acid describedabove, covalently attaching the nucleic acid to the complex, forming a"redox hydrogel" on the surface of the electrode.

In this embodiment, a single stranded nucleic acid probe containing atleast one electron transfer moiety is attached via this redox hydrogelto the surface of an electrode. Hybridization of a target sequence canthen be measured as a function of conductivity between the electrontransfer moiety covalently attached to one end of the nucleic acid andthe electrode at the other end. This may be done using equipment andtechniques well known in the art, such as those described in thereferences cited above.

In similar embodiments, two nucleic acids are utilized as probes asdescribed previously. For example, one nucleic acid is attached to asolid electrode, and the other, with a covalently attached electrontransfer moiety, is free in solution. Upon hybridization of a targetsequence, the two nucleic acids are aligned such that electron transferbetween the electron transfer moiety of the hybridized nucleic acid andthe electrode occurs. The electron transfer is detected as outlinedabove, or by use of amperometric, potentiometric or conductometricelectrochemical sensors using techniques well known in the art.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.

EXAMPLES

The amino-modified monomer units are prepared by variation of publishedprocedures and are incorporated into a growing oligonucleotide bystandard synthetic techniques. The procedure is applicable to both DNAand RNA derivatives.

Example 1 Synthesis of an Oligonucleotide Duplex with Electron TransferMoieties at the 5' Termini

In this example an eight nucleotide double stranded nucleic acid wasproduced, with each single strand having a single electron transfermoiety covalently attached to the 5' terminal uridine nucleotide at the2' carbon of the ribose sugar.

Step 1: Synthesis of 5'-di(p-methoxyphenyl)methylether-2'-(trifluoroacetamido)-2'-deoxyuridine

2'-(trifluoroacetamido)-2'-deoxyuridine (2.0 g, 5.9 mmoles) prepared byminor modification of published procedures (Imazawa, supra) wasrepeatedly dissolved in a minimum of very dry CH₃ CN and rotaryevaporated to dryness and then transferred to inert atmosphere vacuumline and further dried for a period of 1 hour. The following procedurefor the synthesis of the material was adapted from Gait (supra): Underpositive pressure argon, the material was dissolved in freshly dried anddistilled pyridine and with stirring, 0.05 equivalents (wt.) of4-dimethylaminopyridine (DMAP), 1.5 equivalents of triethylamine (TEA)and 1.2 equivalents of 4,4'-dimethoxytrityl chloride (DMTr-Cl) wereadded to the reaction mixture. The progress of the reaction wasmonitored by silica gel TLC (98:2 methylene chloride:methanol, mobilephase). After 30 minutes, an additional 0.5 equivalents each of DMTr-Cland TEA were added and the reaction allowed to proceed for an additionalthree hours. To this reaction mixture was added an equal volume of waterand the solution extracted several times with diethyl ether. The etherlayers were rotary evaporated to dryness, redissolved in a minimumamount of methylene chloride and purified by flash chromatography (99:1methylene chloride:methanol, mobile phase), to obtain the5'-di(p-methoxyphenyl)methylether-2'-(trifluoroacetamido)-2'-deoxyuridineproduct.

Step 2: 5'-2'-aminouridine-GCTACGA and 5'-2'-aminouridine-CGTAGCA

5'-di(p-methoxyphenyl)methylether-2'-(trifluoroacetamido)-2'-deoxyuridine was dried under reducedpressure (glass) and dissolved in freshly dried and distilled CH₃ CN andplaced in a specially made conical vial and placed on an ABI DNAsynthesizer. The program for the preparation of standard (i.e.unmodified) oligonucleotides was altered during the final base(amino-modified) addition to a 15-30 minute coupling time. Theoligonucleotide was cleaved from the column by standard procedures andpurified by C-18 reverse phase HPLC. In this manner5'-2'-aminouridine-GCTACGA and 5'-2'-aminouridine-CGTAGCA were prepared.In addition, unmodified complementary strands to both products were madefor use in the electron transfer moiety synthesis below.

Step 3: 5'-2'-ruthenium bisbipyridineimidazole-aminouridine-GCTACGA

5'-2'-aminouridine GCTACGA produced in the previous step was annealed tothe complementary unmodified strand using standard techniques. Allmanipulations of the annealed duplex, prior to the addition of thetransition metal complex were handled at 4° C. In order to insure thatthe DNA remained annealed during modification, the reactions wereperformed in 1M salt. The 5'-amino modified duplex DNA was dissolved in0.2 M HEPES, 0.8 M NaCl, pH 6.8 and repeatedly evacuated on a Schlenkline. Previously prepared ruthenium bisbipyridine carbonate wasdissolved in the above buffer and oxygen was removed by repeatedevacuation and purging with argon via a Schlenk line. The rutheniumcomplex was transferred to the DNA solution via cannulation(argon/vacuum) and the reaction allowed to proceed under positivepressure argon with stirring for 24 hours. To this reaction, 50equivalents of imidazole was added to the flask and the reaction allowedto proceed for an additional 24 hours. The reaction mixture was removedfrom the vacuum line and applied to a PD-10 gel filtration column andeluted with water to remove excess ruthenium complex. The volume of thecollected fractions was reduced to dryness via a speed vac and the solidtaken up in 0.1 M triethylammonium acetate (TEAC) pH 6.0. The duplex DNAwas heated to 60° C. for 15 minutes with 50% formamide to denature theduplex. The single stranded DNA was purified using a C-18 reverse phaseHPLC column equiped with a diode array detector and employing a gradientfrom 3% to 35% acetonitrile in 0.1 M TEAC, pH 6.0.

Step 4: 5'-2'-ruthenium tetraminepyridine-aminouridine-CGTAGCA

5'-aminouridine-CGTAGCA (0.3 μm) was dissolved in 0.2 M HEPES, 0.8 MNaCl buffer, pH 6.8 and degassed on the vacuum line. To a 10 ml conicalshaped flask equipped with a stirring bar and septum was slurriedRu(III) tetraaminepyridine chloride (10 μm), in the same buffer. In aseparate flask, Zn/Hg amalgam was prepared and dried under reducedpressure and the ruthenium(III) solution transferred (via cannulation)to the Zn/Hg amalgam. The immediate formation of a clear yellow solution(λ_(max) =406 nm) indicated that the reduced form of the ruthenium hadbeen achieved and the reaction allowed to proceed for 30 minutes. Thissolution was transferred to the flask containing the amino-modified DNAand the reaction allowed to proceed at room temperature for 24 hoursunder argon. The reaction mixture was removed from the vacuum line and a50 fold excess of cobalt EDTA (Kirschner, Inorganic Synthesis (1957), pp186) added to the solution. The solution was applied to Sephadex G-25gel filtration column to remove excess ruthenium complex and furtherpurified by reverse phase HPLC as described above. The two rutheniummodified nucleotides were annealed by standard techniques andcharacterized (see Example 5).

Example 2 Synthesis of Long DNA Duplexes with Electron Transfer Moietiesat the 5' Termini

In this example, an in vitro DNA amplification technique, PCR (reviewedin Abramson et al., Curr. Op. in Biotech. 4:41-47 (1993)) is used togenerate modified duplex DNA by polymerization of nucleotides offmodified primer strands (Saiki et al., Science 239:487 (1988)). Twooligonucleotides 18 bases in length and not complementary to each otherare synthesized with amino-modification to the 2'-ribose position of the5' nucleotides, as in example 1.

A series of oligonucleotides of increasing lengths starting at 40 basesare chemically synthesized using standard chemistry. Each of the PCRtemplates shares a 5' sequence identical to one modified 18 mer. The 3'end of the template oligonucleotide shares a sequence complementary tothe other 18mer.

PCR rapidly generates modified duplex DNA by the catalysis of 5'-3' DNAsynthesis off of each of the modified 18 mers using the unmodifiedstrand as a template. One hundred nanomoles of each of the two modified18 mers are mixed in 1 ml of an aqueous solution containing 2,000 unitsof Taq polymerase, deoxyribonucleoside triphosphates at 0.2 M each, 50mM KCl, 10 mM Tris-Cl, pH 8.8, 1.5 mM MgCl₂, 3 mM dithiothreitol and 0.1mg/ml bovine serum albumin. One femtomole of the template strand 40bases in length is added to the mixture. The sample is heated at 94° C.for one minute for denaturation, two minutes at 55° C. for annealing andthree minutes at 72° C. for extension. This cycle is repeated 30 timesusing an automated thermal cycler.

The amplified template sequences with transition metal complexes on both5' termini are purified by agarose gel electrophoresis and used directlyin electron transfer applications.

Example 3 Synthesis of Covalently Bound Electron Transfer Moieties atInternucleotide Linkages of Duplex DNA

In this example, alternative backbones to phophodiester linkages ofoligonucleotides are employed. Functional groups incorporated into theseinternucleotide linkages serve as the site for covalent attachment ofthe electron transfer moieties. These alternate internucleotide linkagesinclude, but are not limited to, peptide bonds, phosphoramidate bonds,phosphorothioate bonds, phosphorodithioate bonds andO-methylphosphoramidate bonds.

The preparation of peptide nucleic acid (PNA) follows literatureprocedures (See Engholm, supra), with the synthesis of Boc-protectedpentaf lurophenyl ester of the chosen base (thymidine). The resultingPNA may be prepared employing Merrifield's solid-phase approach(Merrifield, Science, 232:341 (1986)), using a single coupling protocolwith 0.1 M of the thiminyl monomer in 30% (v/v) DMF in CH₂ Cl₂. Theprogress of the reaction is followed by quantiative ninhydrin analysis(Sarin, Anal. Biochem., 117:147 (1981)). The resulting PNA may bemodified with an appropriate transition metal complex as outlined inexample 1.

The synthesis of phosphoramidate (Beaucage, supra, Letsinger, supra,Sawai, supra) and N-alkylphosphoramidates (Jager, supra) internucleotidelinkages follows standard literature procedures with only slightmodification (the procedures are halted after the addition of a singlebase to the solid support and then cleaved to obtain a dinucleotidephosphoramidate). A typical example is the preparation of the phenylester of 5'O-isobutyloxycarbonylthymidyl-(3'-5')-5'-amino-5'-deoxythymidine(Letsinger, J. Org. Chem., supra). The dimer units are substituted forstandard oligonucleotides at chosen intervals during the preparation ofDNA using established automated techniques. Transition metalmodification of the modified linkages takes place as described inExample 1.

The synthesis of phosphorothioate and phosphorodithioate (Eckstein,supra, and references within) internucleotide linkages is welldocumented. A published protocol utilizes an Applied Biosystems DNAsynthesizer using a modified β-cyanoethylphosphoramidite cycle that capsafter sulphurization with tetraethylthiuram disulfide (TETD) (Iyer, J.Org. Chem. 55:4693 (1990)). The phosphorothioate and phosphorodithioateanalogs are prepared as dimers and cleaved from the solid support andpurified by HPLC (acetonitrile/triethylammonium acetate mobile phase).

Example 4 Synthesis of Two Oligonucleotides Each with an ElectronTransfer oiety at the 5' Terminus

In this example, two oligonucleotides are made which hybridize to asingle target sequence, without intervening sequences. Oneoligonucleotide has an electron donor moiety covalently attached to the5' terminus, and the other has an electron acceptor moiety covalentlyattached to the 5' terminus. In this example, the electron transferspecies are attached via a uradine nucleotide, but one skilled in theart will understand the present methods can be used to modify any of thenucleotides. In addition, one skilled in the art will recognize that theprocedure is not limited to the generation of 8-mers, but is useful inthe generation of oligonucleotide probes of varying lengths.

The procedure is exactly as in Example 1, except that the 8-mersgenerated are not complementary to each other, and instead arecomplementary to a target sequence of 16 nucleotides. Thus the finalannealing step of step 4 of Example 1 is not done. Instead, the twomodified oligonucleotides are annealed to the target sequence, and theresulting complex is characterized as in Example 5.

Example 5 Characterization of Modified Nucleic Acids

Enzymatic Digestion

The modified oligonucleotides of example 1 were subjected to enzymaticdigestion using established protocols and converted to their constituentnucleosides by sequential reaction with phosphodiesterase and alkalinephosphatase. By comparison of the experimentally obtained integratedHPLC profiles and UV-vis spectra of the digested oligonucleotides tostandards (including 2'-aminouridine and 2'-aminoadenine), the presenceof the amino-modified base at the predicted retention time andcharacteristic UV-vis spectra was confirmed. An identical procedure wascarried out on the transition metal modified duplex DNA and assignmentsof constituent nucleosides demonstrated single-site modification at thepredicted site.

Fluorescent Labeled Amino-modified Oligonucleotides

It has been demonstrated that the fluorochrome, fluoresceinisothiocyanate (FITC) is specific for labeling primary amines onmodified oligonucleotides while not bonding to amines or amides presenton nucleotide bases (Haugland, Handbood of Fluorescent Probes andResearch Chemicals, 5th Edition, (1992)). This reaction was carried outusing the amino-oligonucleotide synthesized as described in example 1and on an identical bases sequence without the 2'-amino-ribose grouppresent. Fluorescence spectroscopic measurements were acquired on boththese oligonucleotides and the results confirm the presence of the amineon the 5'-terminal ribose ring.

Thermodynamic Melting Curves of Modified Duplex DNA

A well established technique for measuring thermodynamic parameters ofduplex DNA is the acquisition of DNA melting curves. A series of meltingcurves as a function of concentration of the modified duplex DNA wasmeasured via temperature controlled UV-vis (Hewlett-Packard), usingtechniques well known in the art. These results confirm thathybridization of the amino-modified and transition metal modified DNAhad taken place. In addition, the results indicate that the modified DNAform a stable duplex comparable to the stability of unmodifiedoligonucleotide standards.

Two Dimensional Nuclear Magnetic Resonance (NMR) Spectroscopy

The amino-modified oligonucleotides synthesized as a part of this workwere prepared in sufficient quantities (6 micromoles) to permit theassignment of the ¹ H proton NMR spectra using a 600 MHz Varian NMRspectrometer.

Measurement of the Rate of Electron Transfer

An excellent review of the measurement techniques is found in Winkler etal., Chem. Rev. 92:369-379 (1992). The donor is Ru(bpy)₂ (NHuridine)im,E⁰ ˜1 V, and the acceptor is Ru(NH₃)₄ py(NHuridine)im, E⁰ ˜330 mV. Thepurified transition metal modified oligonucleotides (U_(NHRu)(bpy)2imGCATCGA and U_(NHRu)(NH3)4(py)im CGATGCA were annealed by heating anequal molar mixture of the oligonucleotides (30 μmolar: 60 nmoles of DNAin 2 ml buffer) in pH 6.8 (100 mM NaPi, 900 mM NaCl) to 60° C. for 10minutes and slowly cooling to room temperature over a period of 4 hours.The solution was transferred to an inert atmosphere cuvette equippedwith adapters for attachment to a vacuum line and a magnetic stirringbar. The solution was degassed several times and the sealed apparatusrefilled repeatedly with Ar gas.

The entire apparatus was inserted into a cuvette holder as part of theset-up using the XeCl excimer-pumped dye laser and data acquired atseveral wavelengths including 360, 410, 460 and 480 nm. The photoinducedelectron transfer rate is 1.6×10⁶ s⁻¹ over a distance of 28 Å.

Example 6 Synthesis of a Single Stranded Nucleic Acid Labeled with TwoElectron Transfer Moieties

This example uses the basic procedures described earlier to generate twomodified oligonucleotides each with an electron transfer moietyattached. Ligation of the two modified strands to each other produces adoubly labeled nucleic acid with any of four configurations: 5' and 3'labeled termini, 5' labeled terminus and internal nucleotide label, 3'labeled terminus and internal nucleotide label, and double internalnucleotide labels. Specifically, the synthesis of an oligonucleotide 24bases in length with an electron transfer donor moiety on the 5' end andan internal electron transfer moiety is described.

Five hundred nanomoles of each of two 5'-labeled oligonucleotides 12bases in length are synthesized as detailed above with ruthenium (II)bisbipyridine imidazole on one oligonucleotide, "D" and ruthenium (III)tetraamine pyridine on a second oligonucleotide, "A".

An unmodified oligonucleotide 24 bases in length and complementary tothe juxtaposition of oligonucleotide "D" followed in the 5' to 3'direction by oligonucleotide "A" is produced by standard synthetictechniques. Five hundred nanomoles of this hybridization template isadded to a mixture of oligonucleotides "A" and "D" in 5 ml of an aqueoussolution containing 500 mM Tris-Cl, pH 7.5, 50 mM MgCl₂, 50 mMdithiothreitol and 5 mg/ml gelatin. To promote maximal hybridization oflabeled oligonucleotides to the complementary strand, the mixture isincubated at 60° C. for 10 minutes then cooled slowly at a rate ofapproximately 10° C. per hour to a final temperature of 12° C. Theenzymatic ligation of the two labeled strands is achieved with T4 DNAligase at 12° C. to prevent the ligation and oligomerization of theduplexed DNA to other duplexes (blunt end ligation). Alternatively, E.coli DNA ligase can be used as it does not catalyze blunt end ligation.

One hundred Weiss units of T4 DNA ligase is added to the annealed DNAand adenosine triphosphate is added to a final concentration of 0.5 mM.The reaction which catalyzes the formation of a phosphodiester linkagebetween the 5' terminal phosphate of oligonucleotide "A" and the 3'terminal hydroxyl group of oligonucleotide "D" is allowed to proceed for18 hours at 12° C. The reaction is terminated by heat inactivation ofthe enzyme at 75° C. for 10 minutes. The doubly labeled oligonucleotideis separated from the singly labeled oligonucleotides and thecomplementary unlabeled oligonucleotide by HPLC in the presence of ureaas in the previous examples. The doubly labeled oligonucleotide of thisexample is ideally suited for use as a photoactive gene probe asdetailed below.

Example 7 Use of a Doubly Modified Oligonucleotide with ElectronTransfer Moieties as a Photoactive Probe for Detection of ComplementaryNucleic Acid Sequence

This example utilizes the oligonucleotide 24 mer of example 6 in aunique type of gene-probe assay in which removal of unhybridized probeprior to signal detection is not required. In the assay procedure, aregion of the gag gene of human immunodeficiency virus type I (HIV-I) isamplified by the polymerase chain reaction (Saiki et al., Science239:487-491 (1988)). This region of HIV-I is highly conserved amongdifferent clinical isolates.

The amplified target DNA versus controls lacking in HIV-I DNA are addedto a hybridization solution of 6×SSC (0.9 M NaCl, 0.09 M Na citrate, pH7.2) containing 50 nanomoles of doubly labeled 24 mer probe of example6. Hybridization is allowed to proceed at 60° C. for 10 minutes withgentle agitation. Detection of electron transfer following laserexcitation is carried out as in example 5. Control samples which lackthe hybridized probe show negligible electron transfer rates. Probeshybridized to the gag sequence show efficient and rapid electrontransfer through the DNA double helix, providing a highly specific,homogeneous and automatable HIV-I detection assay.

A similar homogeneous gene probe assay involves the use of two probes,one an electron donor and the other an electron acceptor, whichhybridize with the gag region of HIV-I in a tandem configuration, oneprobe abutting the other. In this assay, electronic coupling between thetwo electron transfer moieties depends entirely on hybridization withthe target DNA. If appropriate, the electron transfer from one probe tothe other is enhanced by the ligation of the juxtaposed ends using T4DNA ligase as in example 6.

What is claimed is:
 1. A single-stranded nucleic acid containing at least one electron donor moiety and at least one electron acceptor moiety, said electron donor moiety and said electron acceptor moiety being covalently attached to said nucleic acid.
 2. A composition comprising a first single stranded nucleic acid comprising an electrode and a second single stranded nucleic acid comprising a covalently attached electron transfer moiety.
 3. A method of detecting a target sequence in a nucleic acid sample comprising:a) applying a first input signal to a hybridization complex comprising said target sequence, which if present, is hybridized to at least a first nucleic acid, wherein said hybridization complex has a covalently attached electron donor moiety and a covalently attached electron acceptor moiety; and b) detecting electron transfer between said electron donor moiety and said electron acceptor moiety as an indication of the presence or absence of said target sequence.
 4. A method according to claim 3 wherein at least one of said electron donor and said electron acceptor moieties is an electrode.
 5. A method according to claim 3 wherein at least one of said electron donor and said electron acceptor moieties is a transition metal complex.
 6. A method according to claim 3 wherein said electron donor moiety is an electrode and said electron acceptor moiety is a transition metal complex.
 7. A method according to claim 3 wherein said first nucleic acid comprises a covalently attached electron donor moiety and a covalently attached electron acceptor moiety.
 8. A method according to claim 3 wherein said first nucleic acid comprises a covalently attached electron donor moiety and said target sequence comprises a covalently attached electron acceptor moiety.
 9. A method according to claim 3 wherein at least one of said electron donor and said electron acceptor moieties is attached to a terminal base.
 10. A method according to claim 3 wherein said nucleic acid comprises a ribose-phosphate backbone.
 11. A method according to claim 10 wherein at least one of said electron donor and said electron acceptor moieties is attached to a ribose of said ribose-phosphate backbone.
 12. A method according to claim 10 wherein at least one of said electron donor and said electron acceptor moieties is attached to a phosphate of said ribose-phosphate backbone.
 13. A method according to claim 3 wherein said nucleic acid is a nucleic acid analog.
 14. A method according to claim 13 wherein said nucleic acid analog is peptide nucleic acid.
 15. A method according to claim 14 wherein said transition metal complex comprises ruthenium, rhenium, osmium, platinum, copper or iron.
 16. A method according to claim 3 wherein at least one of said electron donor and said electron acceptor moieties is an organic electron donor or acceptor. 